2.4 Resting cell bioconversions
The cells were cultivated as described above, harvested by
centrifugation (10 min, 5,000 g, RT), and resuspended to a specific cell
concentration in 100 mM potassium phosphate buffer (pH 7.4) supplemented
with 1 % (w/v) glucose (KPi-g buffer). The cells were transferred to
baffled Erlenmeyer flasks (100 mL) or microcentrifuge tubes (2 mL) with
liquid volumes of 10 or 1 mL, respectively, equilibrated at 30 °C for 10
min (flasks in a water bath at 250 rpm; microcentrifuge tubes in a
ThermoMixer® C (Eppendorf, Hamburg) at 2,000 rpm), then provided with
the substrate (as stated in the Table and Figure legends).
Biotransformations were stopped by the addition of 0.5 mL ice-cold
diethyl ether containing 0.2 mM n-decane as an internal standard to 1 mL
sample. After 2 min extraction by vortexing and short centrifugation,
the organic phase was dried over water-free
Na2SO4 before it was transferred to a GC
vial for analysis. The aqueous phase was removed with a syringe from the
microcentrifuge tube and stored at -20 °C for HPLC analysis.
For the conversion of 5 mM cyclohexane, 250 mL baffled Erlenmeyer flasks
were used with a liquid volume of 40 mL. The caps contained two septa, a
Teflon septum facing the inner side of the flask, and a silicon septum
facing outwards. Pure cyclohexane (21.8 µL) was added to start the
reaction. For each sampling point, 1.5 mL liquid volume was removed
through the septa using a syringe. From this sample, 1 mL was extracted
with diethyl ether for GC analysis as described above and 0.5 mL was
used for HPLC analysis.
For details on analytical methods refer to Supplementary Information
(Section 3).